The present invention relates to a plasmid vector usable in genetic recombinant technology and to a method for expressing a gene by using the plasmid vector. The present invention also relates to a method for isolating a desired gene by using such a plasmid vector. In addition, the present invention relates to a restriction enzyme and a gene thereof available as a genetic engineering reagent, in more detail to the AccIII restriction endonuclease and a DNA coding therefor.
In constructing an expression system for a desired gene by genetic recombinant technology, expression of the gene is controlled by bringing it under control of a promoter recognized by the RNA polymerase of the host used. In the case of a gene encoding a protein harmful to the host, however, plasmid construction itself is sometimes hampered by expression of the product of the gene due to the inability to stringently control the expression of the promoter used.
As an expression system resolving that problem, the pET system (produced by Novagen) has been developed, which uses the RNA polymerase of the bacteriophage T7, which infects Escherichia coli, with Escherichia coli as a host [journal of Molecular Biology, Vol. 189, pp. 113-130 (1986); Gene, Vol. 56, pp. 125-135 (1987)]. The pET system is a system that allows T7 RNA polymerase, which has high promoter recognition specificity and high transcription activity, to be expressed in Escherichia coli, which T7 RNA polymerase transcribes a desired gene placed downstream of the T7 promoter on an expression vector and causes high expression of the gene. Because transcription of the desired gene occurs in the presence of T7 RNA polymerase, plasmid construction in the host is possible without expressing the desired gene, provided that the host does not produce the polymerase; plasmid construction itself is never hampered, as in cases where the expression system is constructed, while the desired gene is kept under control of a promoter recognized by the RNA polymerase of the host.
However, because the T7 RNA polymerase gene has been cloned onto the xcex-phage vector and lysogenized into the expression host, there is no freedom of host choice; painstaking procedures are needed if the host is changed. In addition, because the expression of T7 RNA polymerase in the host is not stringently controlled, T7 RNA polymerase is expressed even when the host is in a non-inductive condition, resulting in expression of the desired gene placed downstream of the T7 promoter on the expression vector even in a non-inductive condition. To suppress such expression of the desired gene in a non-inductive condition, T7 RNA polymerase activity is inhibited using T7 lysozyme, a T7 RNA polymerase inhibitor [Journal of Molecular Biology, Vol. 219, pp. 37-44 (1991)], or T7 RNA polymerase is prevented from getting access to the T7 promoter by placing a lactose operator downstream of the T7 promoter [Journal of Molecular Biology, Vol. 219, pp. 45-59 (1991)].
However, even these countermeasures are unsatisfactory in terms of effect against T7 RNA polymerase of high transcription activity so that the activity of T7 RNA polymerase in a non-inductive condition cannot be completely inhibited. For this reason, if the desired gene product is lethal to the host, it is impossible in some cases to prepare a transformant for expression of the gene, even when plasmid construction is possible. In other words, the pET system involves two problems to be resolved: one of the inability to freely change the host, and the other of inaccurate control of T7 RNA polymerase expression.
On the other hand, there is a bacteriophage having characteristics similar to those of the bacteriophage T7, known as the bacteriophage SP6 [Science, Vol. 133, pp. 2069-2070 (1961)], which infects Salmonella typhimurium. The RNA polymerase produced by the bacteriophage SP6, a single peptide having-a molecular weight of about 100,000, is commonly used for in vitro RNA synthesis since it possesses high promoter recognition specificity and high transcription activity [Journal of Biological Chemistry, Vol. 257, pp. 5772-5778 (1982); Journal of Biological Chemistry, Vol. 257, pp. 5779-5788 (1982)]. In addition, the SP6 RNA polymerase gene has already been cloned and expressed in large amounts in Escherichia coli [Nucleic Acids Research, Vol. 15, pp. 2653-2664 (1987)].
Genes whose expression product acts lethally on hosts are exemplified by restriction endonuclease genes. Essentially, restriction endonucleases are utilized for self-defense by cleaving phages and other exogenous DNA entering the cells of microorganisms that produce the restriction endonucleases. On the other hand, microorganisms that produce restriction endonucleases mostly produce modification enzymes that recognize the same base sequences as those of the restriction endonucleases, to protect their own DNA against cleavage by the restriction endonucleases. Specifically, a modification enzyme modifies DNA by adding a methyl group to one or more bases in the base sequence recognized thereby, to make it impossible for the restriction endonuclease that recognizes the same sequence as that of the modification enzyme to bind thereto or to cleave the DNA. This mechanism is called restriction modification system, and the pair of genes of the restriction endonuclease and modification enzyme that constitute the restriction modification system called restriction modification system gene. Therefore, when the restriction endonuclease gene is expressed in a microorganism lacking a modification enzyme gene from the restriction modification system gene, the microorganism""s DNA is cleaved, resulting in cell death. In fact, there are two modification enzyme genes in the MboI restriction modification system gene; it has been reported that cloning of restriction endonuclease genes is impossible due to incomplete modification of the host DNA in the case of incomplete methylation in the co-presence of either modification enzyme gene alone [Nucleic Acids Research, Vol. 21, pp. 2309-2313 (1993)].
Also, it has been demonstrated that if a restriction modification system gene is lost from a cell retaining the restriction modification system gene, a lack of modification activity in the cell results in incomplete methylation of genomic DNA, which in turn causes lethal cleavage of its own genomic DNA by a very small amount of restriction endonuclease remaining therein [Science, Vol. 267, pp. 897-899 (1995)]. In summary, in the absence of modification enzymes that constitute a restriction modification system, restriction endonucleases behave as proteins very harmful to cells; separate cloning and expression of their genes have been impossible by prior art technologies.
Concerning restriction endonucleases, restriction endonucleases can be classified by their enzymatic properties into three types: I, II and III. Type II restriction endonucleases, in particular, each of which recognizes a particular DNA base sequence and cleaves it at a particular site in or near the sequence, are extensively used in the field of genetic engineering, and restriction endonucleases of this type with various specificities have been isolated from a variety of microorganisms [Nucleic Acids Research, Vol. 24, pp. 223-235 (1996)]. In the present specification, a type II restriction endonuclease is hereinafter referred to as xe2x80x9crestriction endonucleasexe2x80x9d. It should be noted, however, that some microorganisms produce only small amounts of restriction endonuclease, and others produce a plurality of restriction endonucleases. For example, the restriction endonuclease AccIII is produced by Acinetobacter calcoaceticus (hereinafter referred to as Acc bacterium), which has been deposited under accession number FERM BP-935 at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry [address: 1-3, Higashi 1-chome, Yatabemachi, Tsukuba-gun, Ibaraki, 305, Japan] since Nov. 9, 1985 (date of original deposition), but the amount of the enzyme produced is small and this microorganism also produces the restriction endonucleases AccI and AccII simultaneously. Therefore, advanced production technology is needed to provide the restriction endonuclease AccIII as a reagent of high purity and low cost using this microorganism. In providing a restriction endonuclease as a reagent of high purity and low cost, it is effective to isolate the desired restriction endonuclease gene and selectively produce the desired restriction endonuclease in large amounts by genetic engineering technology. To accomplish this purpose, some methods of isolating restriction endonuclease genes have been reported.
First, there may be mentioned the xe2x80x9cshotgunxe2x80x9d method, wherein the genomic DNA of a microorganism that produces a restriction endonuclease is cleaved using the appropriate restriction endonuclease, the resulting fragment is inserted into an appropriate plasmid vector, and a clone expressing the restriction endonuclease gene is selected. Screening methods for desired clones are exemplified by a method wherein a restriction modification system gene is isolated with resistance to phage infection as an index, on the basis of the self-defence function acquired by the host upon introduction of the restriction modification system gene thereinto [PstI: Proceedings of the National Academy of Science of the USA, Vol. 78, pp. 1503-1507 (1981)]. This method, however, necessitates that the size of the restriction modification system gene falls within a range allowing its isolation, and that the expression of the restriction modification system gene isolated exhibits sufficient phage resistance to allow the selective survival of the host. On the other hand, as a general feature of restriction modification system genes, there may be mentioned the close location on the genome of restriction endonuclease genes and modification enzyme genes; in fact, this has been confirmed in many restriction modification system genes that have so far been obtained [Nucleic Acids Research, Vol. 19, pp. 2539-2566 (1991)]. Accordingly, there is a method wherein a restriction modification system gene is screened for with the expression of a modification enzyme gene as an index on the basis of the above-described feature [Japanese Patent Laid-Open No. 63-87982; Nucleic Acids Research, Vol. 19, pp. 1831-1835 (1991)]. When the restriction endonuclease gene is not close to the modification enzyme gene, however, this method fails to yield the restriction endonuclease gene.
Furthermore, the above-described xe2x80x9cshotgunxe2x80x9d method poses a fundamental problem associated with a difference in transcription-translation mechanism between the genomic DNA source organism and the host. For example, in the case of insufficient gene expression due to the failure of the promoter and ribosome binding site accompanying the restriction modification system gene to function well in the host, much labor is needed to select transformants containing the desired gene, even if obtained. To avoid this drawback, there is a method wherein the amino acid sequence of the restriction endonuclease protein is analyzed, the restriction endonuclease gene is obtained from the genomic DNA of a microorganism that produces the restriction endonuclease by PCR-based DNA amplification on the basis of the sequence data obtained, and wherein a known protein expression system is utilized [Japanese Patent Laid-Open No. 6-277070]. Because the presence of a restriction endonuclease is lethal to the host in conventional protein expression systems, there is a need to protect the host by, for example, allowing a modification enzyme that constitutes a restriction modification system together with the enzyme to be co-present.
Although all the above-described methods of the isolation of restriction endonuclease genes necessitate the simultaneous isolation of the restriction endonuclease gene and a modification enzyme gene that constitutes a restriction modification system gene together with the gene, another method of isolating the restriction endonuclease gene alone has been reported [Nucleic Acids Research, Vol. 22, pp. 2399-2403 (1994)]. In that method, however, it is intended to isolate a gene encoding a restriction endonuclease for which optimal temperature for enzyme activity is around 70xc2x0 C.; the co-presence of a modification enzyme gene is necessary when the gene to be isolated encodes a restriction endonuclease showing high specific activity near host culturing temperature.
Exceptionally, there are restriction endonucleases that do not show cleavage activity unless a particular nucleic acid base in their DNA recognition sequence has not been modified by methylation, like the restriction endonuclease DpnI. Genes for restriction endonucleases possessing this property are thought to be exceptional in that they can be isolated even in the absence of another particular gene by selecting the appropriate host organism. In fact, the mrr gene has been isolated, which encodes the Mrr protein, which is not a type II restriction endonuclease but which recognizes a particular DNA base sequence containing a methylated nucleic acid base and exhibits DNA cleavage activity [Journal of Bacteriology, Vol. 173, pp. 5207-5219 (1991)].
As stated above, isolation of a restriction endohuclease gene by the prior art necessitates the simultaneous expression of the gene and a modification enzyme gene that constitutes a restriction modification system gene together with the gene, except for special cases.
Therefore, a first object of the present invention provides a plasmid vector capable of isolating such a gene that an isolation thereof or a construction of an expression system thereof has been difficult in the prior arts because the gene product is lethal or harmful to a host, and capable of introducing into a host to express the protein efficiently. A second object of the present invention provides a method for expressing a desired gene by using this plasmid vector. A third object of the present invention provides a method for isolating a desired gene by using this plasmid vector, especially for isolating a restriction endonuclease gene without co-existence of a modification enzyme gene constituting a restriction modification system. A fourth object of the present invention provides a polypeptide possessing an activity of an AccIII restriction endonuclease. In addition, a fifth object of the present invention provides a DNA which encodes a polypeptide possessing an activity of an AccIII restriction endonuclease.
First, to resolve the problems in the pET system, the present inventors have constructed an expression system using SP6 RNA polymerase as a new accurate expression control expression system, and assessed the system.
Because the expression system is constructed using a system plasmid inserted the SP6 RNA polymerase gene into the miniF plasmid, expression systems for the desired gene can be constructed in various strains of Escherichia coli by simultaneously introducing an expression vector harboring the desired gene cloned downstream of the SP6 promoter and this system plasmid into the host. In addition, using the lac promoter and antisense technology, the present inventors made it possible to exactly control the expression of the SP6 RNA polynerase gene on the system plasmid. Assessing this expression system using the xcex2-galactosidase gene as a reporter gene demonstrated that the expression of the SP6 RNA polymerase gene is accurately controlled in this system, that there is almost no expression of SP6 RNA polymerase in a non-inductive condition, and that induction is followed by the expression of a sufficient amount of SP6 RNA polymerase to efficiently express the desired gene and subsequent expression of the desired gene at a high level.
It was also shown, however, that when the host used is Escherichia coli, the desired gene downstream of the SP6 promoter is expressed in very small amounts even in a non-inductive condition because the SP6 promoter is very weakly but actually recognized by Escherichia coli RNA polymerase. It was thus proven that when the gene product acts very harmfully and lethally to Escherichia coli, expression system construction is impossible so that the object of the present invention cannot be accomplished well.
With these findings in mind, the present inventors have made further extensive investigation and unexpectedly found that (i) expression of the desired gene in a non-inductive condition can be suppressed to undetectable levels, and that (ii) expression induction increases the copy number of the plasmid containing the desired gene and causes RNA polymerase expression, resulting in the transcription and translation of the desired gene placed downstream of a promoter recognized by the RNA polymerase, by using a new system for controlling the expression of the desired gene by means of a combination of two control methods, i.e., control of the copy number of the gene, and transcription control via the promoter. In other words, the plasmid vector of the present invention is a plasmid vector having unique features to resolve the above problems in the field of genetic engineering. The present inventors made further investigation based on this finding, and developed a method for expressing the desired gene using the vector.
The present inventors also have developed a method for isolating a gene whose expression product acts lethally on the host, using the above-described plasmid vector, more specifically a method of isolating a restriction endonuclease gene without the drawbacks of the prior art.
The present inventors also found it possible to isolate DNA encoding the AccIII restriction endonuclease, which had not been obtained so far, without the co-presence of an AccIII modification enzyme, and express the AccIII restriction endonuclease in large amounts, using the above-described method.
(1) The gist of the present invention is concerned with:
A plasmid vector characterized by comprising a promoter sequence to control an expression of a desired gene, the promoter sequence being recognized by an RNA polymerase not inherent to a host, and a replication origin for increasing a copy number by induction with an exogenous factor;
(2) The plasmid vector described in item (1) above, wherein the promoter sequence is recognized by RNA polymerases derived from bacteriophages;
(3) The plasmid vector described in item (2) above, wherein the promoter sequence is recognized by an RNA polymerase derived from SP6 phage;
(4) The plasmid vector described in item (3) above, wherein the promoter sequence contains the base sequence of SEQ ID NO:30 in the Sequence Listing;
(5) The plasmid vector described in any one of items (1) to (4) above, wherein the replication origin is under control of a promoter;
(6) The plasmid vector described in any one of items (1) to (5) above, wherein the replication origin is under control of the lac promoter;
(7) The plasmid vector described in any one of items (1) to (6) above, comprising a drug resistance gene as a selection marker;
(8) The plasmid vector described in item (7) above, which is selected from pACE601, pACE611, pACE701 and pACE702;
(9) A plasmid vector in which a desired gene to be expressed is incorporated into the plasmid vector described in any one of items (1) to (8) above;
(10) A method for expressing a desired gene, characterized by introducing into a host a plasmid vector in which the desired gene is incorporated into the plasmid vector described in any one of items (1) to (8) above, and an RNA polymerase gene which recognizes a promoter sequence in the plasmid vector, and inducing an increase in a copy number of the plasmid vector and an expression of the RNA polymerase by using an exogenous factor to transcribe and translate the desired gene;
(11) The method for expressing a desired gene described in item (10) above, characterized in that the increase in the copy number of the plasmid vector and the expression of the RNA polymerase are induced by respective exogenous factors;
(12) The method for expressing a desired gene described in item (10) above, characterized in that the increase in the copy number of the plasmid vector and the expression of the RNA polymerase are induced by a same exogenous factor;
(13) The method for expressing a desired gene described in any one of items (10) to (12) above, wherein the exogenous factor which induces the increase in the copy number of the plasmid vector, is one or more selected from the group consisting of an addition of isopropyl-xcex2-D-thiogalactoside (IPTG), an addition of lactose, an addition of galactose, an addition of arabinose, a reduction of a tryptophane concentration and an adjustment of a transformant cultivation temperature;
(14) The method for expressing the desired gene described in any one of items (10) to (12) above, wherein the exogenous factor which induces the expression of the RNA polymerase, is one or more selected from the group consisting of an addition of isopropyl-xcex2-D-thiogalactoside (IPTG), an addition of lactose, an addition of galactose, an addition of arabinose, a reduction of a tryptophane concentration and an adjustment of a transformant cultivation temperature;
(15) The method for expressing a desired gene described in item (10) above, characterized in that the RNA polymerase gene is introduced into the host by the other plasmid vector or a phage vector;
(16) The method for expressing a desired gene described in item (10) above, characterized in that the RNA polymerase gene is incorporated into a chromosome of the host;
(17) The method for expressing a desired gene described in item (15) or item (16) above, characterized in that the RNA polymerase gene is derived from SP6 phage;
(18) The method for expressing a desired gene described in any one of items (10) to (17) above, wherein the desired gene encodes a protein lethal or harmful to the host;
(19) The method for expressing a desired gene described in any one of items (10) to (18) above, characterized in that Escherichia coli is used as the host;
(20) A method for isolating a desired gene, characterized in that the plasmid vector described in any one of items (1) to (8) above is employed in the method for isolating the desired gene;
(21) The method for isolating a desired gene described in item (20) above, wherein the desired gene encodes a protein lethal or harmful to a host;
(22) The method for isolating a desired gene described in item (21) above, wherein the gene encoding a protein lethal or harmful to the host is a restriction endonuclease gene;
(23) A polypeptide containing the entire or a portion of the amino acid sequence shown by SEQ ID NO:1 in the Sequence Listing, and possessing an activity of AccIII restriction endonuclease;
(24) A polypeptide having an amino acid sequence resulting from at least one of deletion, addition, insertion or substitution of one or more amino acid residues in the amino acid sequence of SEQ ID NO:1 in the Sequence Listing or a portion thereof, and possessing an activity of AccIII restriction endonuclease;
(25) A DNA encoding a polypeptide which contains the entire or a portion of the amino acid sequence shown by SEQ ID NO:1 in the Sequence Listing, and possesses an activity of AccIII restriction endonuclease;
(26) A DNA containing the entire or a portion of the DNA shown by SEQ ID NO:2 in the Sequence Listing wherein an expression product of the DNA possesses an activity of AccIII restriction endonuclease;
(27) A DNA encoding a polypeptide resulting from at least one of deletion, addition, insertion or substitution of one or more amino acid residues in the amino acid sequence of SEQ ID NO:1 in the Sequence Listing or a portion thereof, and possessing an activity of AccIII restriction endonuclease; and
(28) A DNA capable of hybridizing to the DNA described in any one of items (25) to (27) above, and encoding a polypeptide possessing an activity of AccIII restriction endonuclease.